A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.


Introduction
Photorhabdus luminescens is an entomopathogenic bacterium that has the ability to kill a wide range of insects [1,2]. This bacterium kills the insects by secreting various toxins, such as the toxin complexes (Tcs) and the "makes caterpillars floppy" (Mcf) toxins [1,2]. Photorhabdus insect-related toxins A and B (PirA and PirB) are one of the insecticidal toxins secreted by P. luminescens [2]. In addition to P. luminescens, the relevant genes have also been found in many other bacteria, including Xenorhabdus spps. and Vibrio spps. Notably, the Vibrio parahaemolyticus PirA/PirB homologs (referred to here as PirA vp and PirB vp ) were found to be the key pathogenetic factors of acute hepatopancreatic necrosis disease (AHPND) in shrimps [3]. We note that both insecticidal P. luminescens and marine pathogenic V. parahaemolyticus have their own pirA/pirB genes and that this suggests the functional importance of this toxin family during evolution. However, despite their evident importance, there are still relatively few studies on the PirA/PirB toxins, especially on their cytotoxic mechanisms. In this review, we summarize our current understanding of this toxin family in light of the findings provided by recent reports. We also identify topics that we feel deserve particular attention and hope that this will be helpful for future studies.

Insecticidal PirA/PirB
Photorhabdus PirA/PirB toxins were first determined from the P. luminescens W14 genome [1]. In Photorhabdus, Sodalis, Xenorhabdus, and Yersinia spps., the 2 toxins are encoded by 2 distinct, but adjacent loci on the bacterial genome (Table 1). These pirA/pirB genes are both on the same operon, suggesting that they are co-regulated and co-transcribed. Furthermore, a comparison of the genomic organization of pirA/pirB operons indicated that some phage genes are found in the Yersinia Pir locus and that there is a Fis-type DNA recombinase adjacent to the Photorhabdus operons [15]. This suggests that these pirA/pirB genes have been acquired by horizontal transmission [15].
It was initially proposed that Photorhabdus PirA/PirB was an insect JHE (juvenile hormone esterase)-like protein that disrupted insect metamorphosis by catalyzing the hydrolysis of juvenile hormone (JH) [5]. However, a subsequent study found that Photorhabdus PirA/PirB did not have JHE enzymatic activity [4]. Amino acid sequence analysis then suggested that . Full toxicity is also thought to require the presence of both PirA and PirB [3]. Thus, in an assay of X. nematophila PirA/PirB toxicity to the caterpillar of wax moth Galleria mellonella, the injection of either PirA or PirB alone only resulted in low mortality rates compared to the injection of both PirA and PirB in combination [6]. All of these results support the conclusion that PirA/PirB toxins complement each other to maximize their insecticidal activity. In 2013, although there were no previous reports suggesting that V. parahaemolyticus could cause such severe epidemics in shrimps, specific strains of this halophilic, gram-negative bacterium were identified as the pathogenic source of AHPND [21]. Until that time, V. parahaemolyticus was known primarily for being an opportunistic pathogen that causes human gastroenteritis and other symptoms after the consumption of insufficiently fresh seafood [28]. In 2014, Yang and colleagues used a high-throughput next-generation sequencing (NGS) platform to compare the differences in AHPND pathogenic and nonpathogenic V. parahaemolyticus genome sequences [29]. Surprisingly, the pathogenic genes were not found in the chromosome, and it was shown instead that all the pathogenic V. parahaemolyticus strains contained a plasmid, pVA1, with an approximate size of 70 kb In addition to V. parahaemolyticus, pirA vp /pirB vp like-genes have also been found in other Vibrio spps. [9-13]. However, unlike Photorhabdus and Xenorhabdus, Vibrio pirA/pirB genes were usually found in the plasmid, not in the genomic DNA [9-13]. As shown in Table 1, 5 different Vibrio species have so far been confirmed to contain pirA/pirB genes in their plasmids. Several studies have revealed that different Vibrio species may transfer pVA1-like plasmid from an AHPND-causative strain to a non-AHPND-causative strain through horizontal transfer [31 -34]. It should also be noted that transposase genes are usually found around the Vibrio pirA/pirB genes [3,9,22, [35][36]. For example, in the pVA1 plasmid isolated from V. parahaemolyticus strain 3HP, the pirA/pirB-containing gene cluster (ORF 49-54) is flanked by 2 transposase genes in opposite directions (ORF 48 and ORF 55) [3]. The inverted repeats in the terminals of these 2 transposase genes suggest that the pirA vp /pirB vp gene cluster came from a transposon-mediated horizontal transfer. Similarly, 2 transposase genes were found at both ends of the pirA vp /pirB vp gene cluster in the pVH plasmid from AHPND-causing V. owensii [9]. Transposon genes can also interfere with the expression of pirA vp /pirB vp . Thus in the pVA1-like plasmid isolated from V. parahaemolyticus strain XN87, an out-of-frame insertion caused by a transposon gene fragment was found to prevent the expression of the downstream pirA vp /pirB vp gene [35]. The pirA vp /pirB vp expression was also prevented in another pVA1-like plasmid from a non-AHPND V. parahaemolyticus strain (R14), which has an inserted transposon gene in the pirA vp /pirB vp promoter region [36]. All of these examples demonstrate the importance of transposase in the transfer, deletion, and mutation of the pir-A vp /pirB vp -containing gene cluster in Vibrio spps.

Structural similarities indicate that PirA/PirB toxins are Cry-like pore-forming toxins
V. parahaemolyticus PirA vp /PirB vp were the first structures to be determined from this toxin family [3]. In 2022, the structures of insecticidal PirA/PirB (Photorhabdus akhursti PirA/PirB; PirA pa /PirB pa ) have also been released to the protein data bank (PDB DOI: 10.2210/pdb7FCN/ pdb and 10.2210/pdb7FDP/pdb). Although there are some differences in the amino acid sequences, a comparison of the structures of PirA vp to PirA pa and PirB vp to PirB pa show that they are highly similar (Fig 1A and 1B). We interpret this to mean that during their evolution, these toxins remained functionally conserved.
Bacillus Cry-like toxins belong to a family of insecticidal toxins that kill insects by forming pores on the membrane of midgut cells [37-39], so the structural similarity to Bacillus thuringiensis Cry toxins further suggests that PirA/PirB toxins are also pore-forming toxins [3,17,22]. Structural analysis reveals that the cytotoxic mechanism of the Cry toxins is achieved by 3 functional domains. From the N-terminal to the C-terminal, Cry domain I has pore-forming activity, domain II is used for receptor binding, and domain III is for sugar recognition [37-39]. These domains implement the cytotoxic mechanism of Cry as follows: when Cry toxins reach the midguts of insect larvae, Cry domain III binds to the sugars (e.g., N-Acetylgalactosamine, GalNAc) on the target receptors (e.g., aminopeptidase N, APN; alkaline phosphatase, ALP; cadherin-like receptor, CAD). Domain II then further binds to the target receptors, until finally, the protease activity of the toxin-bound receptors mediates a proteolytic cleavage of the α1 helix of Cry domain I. This cleavage triggers a conformational change of Cry toxin to an active pore-forming state. A subsequent oligomeric form of Cry toxin results in uncontrolled pores on the cell membrane, which in turn leads to cell death by osmotic lysis [37][38][39].
Structural comparisons show that PirA toxins have structural features that are similar to Cry domain III, while the N-terminal and C-terminal of PirB toxins are respectively similar to Cry domains I and II (Fig 1B) [3,17,40]. A reasonable sequence for the cytotoxic mechanism of PirA/PirB would therefore be: first, the formation of a complex that contains the full set of functional domains; the next step would be binding to the respective sugar/receptor on the cell surface; and finally, this would trigger a conformational change that activates the transmembrane activity of PirB, leading to the formation of a higher order oligomer that creates uncontrolled pores.

The expression control, activation, and cytotoxic mechanism of PirA/PirB toxins
While the mechanisms described above provide a plausible outline of how the PirA/PirB toxins might effectively act as Bacillus Cry-like, pore-forming toxins, a more detailed understanding of the toxins' expression control, activation, and cytotoxic mechanisms still needs to be elucidated. Among the various PirA/PirB toxins, the cellular mechanisms of AHPND-causing PirA vp /PirB vp are relatively well studied, so in this section, we summarize these reports and propose various respective mechanisms.

The activation and expression control of PirA vp /PirB vp toxins
Outbreaks of AHPND are known to be influenced by environmental stressors like temperature, pH, and water quality [41][42][43]. There are also reports that the expression of the pirA vp / pirB vp genes is directly affected by environmental changes. For example, pirA vp /pirB vp gene expression was increased when the temperature shifted from a higher to a lower range (26˚-32˚C to 22˚-28˚C) [44]. Apart from water temperature, salinity also plays a key role in regulating the metabolism and virulence gene expression in V. parahaemolyticus [45,46], with the gene expression levels of pirA vp found to be up-regulated when salinity increased significantly. Since its expression is affected by salinity, it has been proposed that transcription of the pirA vp gene might be Na + dependent [47]. The presence of bile acids, which are commonly used as a feed additive for shrimps, was also reported to induce PirA vp /PirB vp toxin release and biofilm formation in AHPND-causing V. parahaemolyticus [23, 48].
Another recent report further suggested that the mRNA and protein expressions of pirA vp / pirB vp were related to the quorum sensing (QS) system and low pH [49]. QS is a cell densitydependent process that is seen in many bacteria, including V. harveyi, V. cholerae, and V. parahaemolyticus [50]. The system is involved in many physiological processes that respond to environmental stress, such as biofilm formation, bioluminescence, conjugation, plasmid transfer, antibiotic production, cell mobility, and sporulation [51]. Notably, the QS system also regulates the expression of virulence factors, such as the type III secretion systems, type VI secretion systems, and the thermostable direct hemolysin genes tdh1 and tdh2 [52-54]. Lin and colleagues showed that an important regulator of the QS system, LuxO vp , is a negative regulator of the pirA vp and pirB vp toxin genes [49]. In addition, a transcription factor in the QS system, AphB vp , was confirmed to bind with the predicted promoter region of pirA vp /pirB vp [49]. In V. cholerae and V. alginolyticus, AphB has been shown to be a key virulence regulator [55,56], and in V. cholerae, downstream genes regulated by AphB are also important for bacterial survival in low pH conditions and anaerobic environments [57]. If AphB is able to respond to environmental changes by altering its DNA-binding ability to regulate these downstream genes, it follows that the regulation of pirA vp /pirB vp genes by AphB vp may also be pH dependent.
It seems clear that environmental factors, especially environmental pH, play important roles in the control of pirA vp /pirB vp gene expression. It would be interesting to investigate the extent to which the gene/protein expression of insecticidal PirA/PirB might similarly be affected by environmental factors, but this has not yet been explored.

The cytotoxic mechanism of PirA vp and PirB vp toxins
Further to the suggestion in Section 4 that PirA/PirB toxins might act as Cry toxins, we now further consider details of the Cry-like cytotoxic mechanisms that might be used by the PirA vp and PirB vp toxins. We have to state here, however, that although we used Cry toxin as a model to propose a mechanism for PirA vp /PirB vp , we cannot be sure that the mechanism of PirA vp / PirB vp is exactly the same as Cry toxin because the research is still in progress. Nevertheless, based on the available informationAU : PleasecheckwhetherthechangesmadeinthesentenceNevertheless; , we believe that the most suitable reference model for Pir-A vp /PirB vp so far is Cry toxin, and that it provides a useful guide to future experiments and thinking.

Forming a Cry-like three-domain toxin.
To begin with, the 2 toxins need to be capable of assembly into a Cry-like, three-domain conformation. In the case of the PirA vp / PirB vp toxins, this kind of physiological binding is supported by several reports [3,40]. Specifically, in 2019, Lin and colleagues determined that PirA vp and PirB vp formed a hetero-tetramer in a 1:1 binding stoichiometry by using gel filtration and densitometric analysis and further proposed a PirA vp /PirB vp hetero-tetramer model by using hydrogen/deuterium exchange mass spectrometry [40]. In this model, the PirA vp /PirB vp complex contains 2 copies of the Cry-like three-domain toxin that supports the pore-forming activity. Interestingly, the binding affinity between PirA vp and PirB vp in solution is not strong, but only moderate to weak (approximately 7 μm, as measured by isothermal titration calorimetry) [40]. This would lead to an instability in the PirA vp /PirB vp complex, but since the above experiments were carried out in the absence of membrane receptors or sugars, it is possible that an interaction with the receptor/sugar itself may be necessary to improve the stability of the PirA vp /PirB vp complex. Another possibility is that this binding instability of the PirA vp /PirB vp complex may be a control mechanism that reduces their combined cytotoxicity by complex dissociation. Although several details still need to be explored, it remains clear that PirA vp and PirB vp do indeed have the ability to form a Cry-like three-domain toxin before binding to their receptors.

Binding to sugars/receptors on the cell membrane.
After forming a Cry-like toxin, PirA/PirB would be expected to target its respective sugars/receptors to attach to the cell membrane. In 2021, Luangtrakul and colleagues reported that shrimp aminopeptidase N receptors are responsible for binding PirA vp and PirB vp . In hemocytes of L. vannamei, 2 aminopeptidase N (APN) proteins were identified, LvAPN1 and LvAPN2, with the membranebound LvAPN1 being shown to act as a receptor for the PirAB vp toxins [58]. APN, which is a membrane protein found in many species, is an enzyme with broad substrate specificity that can remove the N-terminal amino acid from oligopeptides and proteins [59], and it has previously been noted that the binding between Cry toxin and APN in an insect's midgut plays an important role in Cry toxin activation [38,39]. Luangtrakul and colleagues found not only that the expression of LvAPN1 was increased significantly after treatment with AHPND-causing V. parahaemolyticus or PirA vp /PirB vp toxins, but also that truncated LvAPN1 recombinant protein interacted with recombinant PirA vp and PirB vp toxins in a dose-dependent manner [58]. Furthermore, in LvAPN1 knockdown shrimps, toxin-induced mortality, clinical symptoms in the hepatopancreas, and the number of AHPND-causing V. parahaemolyticus bacteria were all reduced [58]. Interestingly, when PirAB vp toxins were used to challenge the LvAPN1-knockdown shrimp, PirB vp toxin was found to be distributed only on the membrane of hemocytes and could not be internalized into the cytoplasm. Another report showed that recombinant PirB vp toxin could interact with histones, resulting in the dephosphorylation of Serine 10 in histone H3, and the subsequent induction of apoptosis [60]. Together, these results suggest that PirB vp toxins may translocate into the cell through direct interaction with LvAPN1, after which they further enter the nucleus and cause hemocyte damage.
In addition to LvAPN1, alpha amylase-like protein was also reported as a potential receptor for PirB vp [61]. Functionally, alpha amylase-like protein has 1,4-α-D-glucan glucanohydrolase activity, and it was identified as a receptor for B. thuringiensis ssp Cry4Ba and Cry11Aa toxins [62]. Two other glycoproteins, beta-hexosaminidase subunit beta and mucin-like 5 AC, which are expressed in the epithelial cells of the shrimp's hepatopancreas, were also found to interact with PirB vp [63]. These studies all strengthen the hypothesis that PirB vp has receptor-binding ability. In the future, site mutation could be used to further identify which regions of PirB vp (e.g., the loop regions of C-terminal domain of PirB vp ) might mediate receptor binding. Meanwhile, we also note that there are slight differences in the receptor binding region of the C-terminal of PirB vp and PirB pa (Fig 1B). This might mean that the target receptors of PirB pa may not be identical to those of PirB vp , but it is more likely to reflect the fact that the same PirA/ PirB receptors in insects and shrimps have evolved into slightly different structures, so that different interfaces are required for efficient binding to these homologous receptors.
Different from the Cry toxins, PirB vp has been found to interact with a fatty acid binding protein (FABP) of L. vannamei directly. Knocking down of LvFABP reduced shrimp mortality and histopathological signs of AHPND, as well as the number of the AHPND-causing V. parahaemolyticus in theAU : PleasecheckifthewordVPAHPNDinthesentenceDifferentfromtheCrytoxins:::shouldbecapt challenged shrimp [64]. FABPs may occur as membrane associated or cytosolic proteins, whereas the cell localization of LvFABP is still unclear. Therefore, it remains to be further investigated whether LvFABP can be a receptor of PirA vp /PirB vp toxin [64].
Compared to its ability to bind to receptors, the ability of PirA/PirB to bind to sugars is not so clear. To date, even though structural similarities to Cry domain III suggest that PirA vp may function as a sugar-binding protein [3,17], this has still not been confirmed experimentally. Interestingly, a recent report indicated that PirB vp , but not PirA vp , is able to target glycosaminoglycans like GalNH 2 and GlcNH 2 [65], suggesting that PirB vp is able to bind both receptors and sugars. This putative sugar-binding ability of PirB vp has also been indirectly confirmed by the binding between PirB vp and glycoproteins [63]. We note, however, that the sugar-binding activity evidently does not play a critical role for the interaction between LvAPN1 and PirA vp / PirB vp , since the LvAPN1 used for the ELISA assay was prepared from E. coli and was not glycosylated [58]. Taken together, the functional roles of PirA vp , as well as the sugar-binding abilities of PirA vp /PirB vp , remain to be further investigated.

Forming transmembrane pores by oligomerization.
The final stage of the cytotoxic mechanism of a pore-forming toxin is to create unregulated pores in the cell membrane. After attaching to the cell membrane by binding to a receptor, pore-forming toxins undergo a conformational change to expose their hydrophobic regions. The resulting toxin is no longer water soluble, but instead becomes an oligomeric transmembrane pore that inserts into the cell membrane [66]. These conformational changes and the oligomerization of pore-forming toxins have been widely studied [66-69]. For example, structural studies of Cytolysin A (ClyA), which is a pore-forming toxin found in several enterobacteria, have revealed both water-soluble monomeric and pore-forming dodecameric conformations [70][71][72].
Unfortunately, however, transmembrane pore conformation is not easily investigated, and we do not yet have a good understanding of the transmembrane pore conformation and oligomerization of PirA vp /PirB vp . Over the past decade, although structural approaches including X-ray crystallography and CryoEM have been used to reveal the transmembrane pore conformations of toxins such as ClyA and Aerolysin [72,73], the currently reported structures for PirA vp /PirB vp were all derived from their water-soluble forms, not their transmembrane forms [3,40]. Furthermore, although PirA vp /PirB vp toxins are able to form a hetero-tetramer in solution [40], this does not reflect the real conformation of the toxins after binding to the membrane.
To investigate the transmembrane stage of novel pore-forming toxins like PirA vp /PirB vp , 2 main challenges need to be overcome. The first challenge is to determine the activation conditions. In the case of PirA vp /PirB vp 's reference, i.e., the Cry toxins, activation is triggered by the proteolytic cleavage of the N-terminal peptide of domain I [38,39], which causes the activated Cry toxin to reconfigure into a transmembrane state. However, the process that initiates this mechanism for PirA vp /PirB vp remains to be elucidated. The second challenge is to create a suitable membrane-like platform for PirA vp /PirB vp to bind and insert into. For this purpose, detergents have often been used to mimic a membranous environment. For example, 0.1% n-Dodecyl-beta-Maltoside (DDM) was used to induce the formation of 13-mers of the E. coli Cytotoxin ClyA [70]. Determining the state of PirA vp /PirB vp in different detergents will be an essential first step in this research.
Our current understanding of these mechanisms therefore suggests a possible cytotoxic mechanism for the PirA/PirB toxins, and particularly for PirA vp /PirB vp , as summarized in Fig 2. Details of the sugar-binding and pore-forming mechanisms will need to be further investigated, but we also note that future PirA vp /PirB vp studies could in turn provide a useful research reference for insecticidal PirA/PirB.

Concluding remarks
As this review has shown, our current understanding of the PirA/PirB toxins is derived from only a limited number of studies, and many mechanisms remain to be elucidated. Meanwhile, these toxins are already deployed in a range of agricultural applications, and further research on this toxin family will be important not just for these insecticidal toxins (e.g., P. luminescens PirA/PirB), but also for potential applications in the prevention and treatment of the shrimp disease AHPND. We hope that this review will help to encourage future study and increase our knowledge of this under-researched toxin family.